Synthetic variants of mycolactone bind and activate Wiskott-Aldrich syndrome proteins

Article abstract of DOI:10.1021/jm5008819

Mycolactone is a complex macrolide toxin produced by Mycobacterium ulcerans, the causative agent of skin lesions called Buruli ulcers. Mycolactone-mediated activation of neural (N) Wiskott-Aldrich syndrome proteins (WASP) induces defects in cell adhesion underpinning cytotoxicity and disease pathogenesis. We describe the chemical synthesis of 23 novel mycolactone analogues that differ in structure and modular assembly of the lactone core with its northern and southern polyketide side chains. The lactone core linked to southern chain was the minimal structure binding N-WASP and hematopoietic homolog WASP, where the number and configuration of hydroxyl groups on the acyl side chain impacted the degree of binding. A fluorescent derivative of this compound showed time-dependent accumulation in target cells. Furthermore, a simplified version of mycolactone mimicked the natural toxin for activation of WASP in vitro and induced comparable alterations of epithelial cell adhesion. Therefore, it constitutes a structural and functional surrogate of mycolactone for WASP/N-WASP-dependent effects.

Full text of DOI:10.1021/jm5008819

Article
pubs.acs.org/jmc
Synthetic Variants of Mycolactone Bind and Activate Wiskott−
Aldrich Syndrome Proteins
Anne-Caroline Chany,*,† Romain Veyron-Churlet,‡ Cedric Tresse,† Veronique Mayau,‡
́
́
Virginie Casarotto,† Fabien Le Chevalier,‡ Laure Guenin-Mace,‡ Caroline Demangel,‡
́
and Nicolas Blanchard*,§
†Laboratoire de Chimie Organique et Bioorganique, Universite
Cedex, France
́
de Haute-Alsace, ENSCMu, 3 Rue A. Werner, 68093 Mulhouse
‡Unite
§Laboratoire de Chimie Molec
Materiaux, 25 Rue Becquerel, 67087 Strasbourg, France
́
d’Immunobiologie de l’Infection, Institut Pasteur, CNRS URA1961, 25 Rue du Dr. Roux, 75724 Paris, France
̀
ulaire, Universite de Strasbourg, CNRS UMR 7509, Ecole Europeenne de Chimie, Polymeres et
́
́
́
́
S
* Supporting Information
ABSTRACT: Mycolactone is a complex macrolide toxin
produced by Mycobacterium ulcerans, the causative agent of
skin lesions called Buruli ulcers. Mycolactone-mediated
activation of neural (N) Wiskott−Aldrich syndrome proteins
(WASP) induces defects in cell adhesion underpinning
cytotoxicity and disease pathogenesis. We describe the
chemical synthesis of 23 novel mycolactone analogues that
differ in structure and modular assembly of the lactone core
with its northern and southern polyketide side chains. The
lactone core linked to southern chain was the minimal
structure binding N-WASP and hematopoietic homolog
WASP, where the number and configuration of hydroxyl
groups on the acyl side chain impacted the degree of binding.
A fluorescent derivative of this compound showed time-dependent accumulation in target cells. Furthermore, a simplified version
of mycolactone mimicked the natural toxin for activation of WASP in vitro and induced comparable alterations of epithelial cell
adhesion. Therefore, it constitutes a structural and functional surrogate of mycolactone for WASP/N-WASP-dependent effects.
INTRODUCTION
■
Mycobacterium ulcerans infection causes a necrotizing skin
disease called Buruli ulcer (BU), which affects a wide range of
hosts including mammals, fishes, and frogs.1−5 Although first
clinically diagnosed in man in 1948 by Australian researchers,6
it was only in 1999 that the exotoxin secreted by the causative
organism, Mycobacterium ulcerans, was discovered by Small.7,8
Extensive multinuclear NMR experiments combined with
chemical synthesis by Kishi9−12 finally led to the full
characterization of a macrolide, named mycolactone A/B
(Figure 1), which consists of a 8-undecenolide (C1−C11
fragment) substituted at C11 by a nine-carbon atom chain
(C12−C20 northern fragment) and at C5 by a pentaenoic acid
ester (C1′-C16′ southern fragment).
The production of mycolactone is unique to M. ulcerans
among human pathogens and underpins bacterial virulence. It
is sufficient to induce Buruli ulcer (BU)-like lesions that are
marked by an intriguing combination of tissue necrosis and lack
of inflammation.13 Neural (N) Wiskott−Aldrich syndrome
proteins (WASPs) is a key player in the ulcerative effects of
mycolactone.14 Unlike its hematopoietic homolog WASP, N-
WASP is ubiquitously expressed. Both proteins transduce
Figure 1. Chemical structure of mycolactone A/B, the exotoxin of M.
ulcerans, as a Z-Δ4′,5′/E-Δ4′,5′ = 60:40 dynamic equilibrium.
endogenous signals into dynamic remodeling of the actin
cytoskeleton via interaction of their C-terminal verprolin-
cofilin-acidic (VCA) domain with the Arp2/3 actin-nucleating
complex.15 Mycolactone was shown to bind WASP and N-
WASP selectively with 20−70 nM affinity, respectively. Binding
triggered transition from an autoinhibited state, in which the
VCA domain is sequestered, into an active state where released
VCA can bind Arp2/3. In epithelial cells, mycolactone-induced
Received: June 10, 2014
Published: August 26, 2014
© 2014 American Chemical Society
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a
Figure 2. Mycolactone analogues by Kishi, Altmann, and Blanchard. Footnote letter in the figure indicates the following: Percentage of cell
rounding after 48 h of incubation with 10 μM of product.33
stimulation of N-WASP resulted in loss of adhesion and
eventually cell death by anoikis. In vivo, injection of
mycolactone into the ears of mice altered the adhesion of
keratinocytes, leading to the rupture of the epidermis.
Mycolactone-induced defects in cell adhesion and skin
ulceration were efficiently suppressed by the N-WASP inhibitor
wiskostatin, demonstrating the critical role of N-WASP in these
processes.
will be needed to determine if WASP/N-WASP or a distinct
molecular receptor mediates the immunomodulatory properties
of mycolactone. Finally, mycolactone was recently reported to
stimulate angiotensin II receptor (AT2R) pathways in
neurons.24 It will be interesting to determine if this new
feature of mycolactone relates to the activation of neural (N)
WASP and resists the inhibitory effect of mycolactone on
membrane protein expression.25
In addition to killing anchorage-dependent cells, mycolac-
tone inhibits at noncytotoxic doses the capacity of immune cells
to express membrane and secreted proteins.16−23 Intriguingly,
this function was efficiently blocked by wiskostatin,21
suggesting that Arp2/3-mediated polymerization of actin is
involved, but wiskostatin was ineffective at rescuing cytokine
production by mycolactone-treated macrophages. Further work
The chemical synthesis of natural products and their
derivatives allows the biological exploration of complex
systems, and programs centered on the synthesis and structural
modulation of mycolactone A/B have been reported by Kishi,
Altmann, and us (Figure 2).2 Kishi clearly opened the way to
chemical modifications of this exotoxin with several generations
of elegant total syntheses of mycolactone A/B and also of the
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Scheme 1. General Synthetic Scheme for the Southern Fragments 18a−c and ent-18a,b of Mycolactone Analogues
other eight naturally occurring mycolactones that differ only by
the nature of the southern fragment.2,9,26−30 However, the
cytopathic effect (CPE, the concentration of mycolactone
analogue for which 90% of the cells round up) of only the
single analogue 1a was disclosed up to now (Figure 2A).9 The
CPE of 1a is 30 nM, which compared very favorably with the
one reported for mycolactone A/B (in the 10 nM range),
showing that a longer southern fragment is well tolerated.
On the basis of Kishi seminal investigations, Altmann and
Pluschke reported in 2013 a structure−activity relationship
study of eight mycolactone A/B analogues, belonging to three
families (Figure 2A).31,32 Instead of measuring CPE, Altmann
and Pluschke used LC50, the concentration of analogue for
which half of the cells were killed, as a measure of activity, thus
allowing a direct (but not strict) comparison of cytotoxic
effects. The first family comprises three different esters of the
C1−C20 fragment of mycolactone A/B (1b−d) in order to
study the influence of the southern chain. The second family is
an incomplete mycolactone A/B, whose northern fragment has
been excised (2a,b). The C5-hydroxy group is either free (2a)
or esterified with sorbic acid (2b). These two families present
either no or a significantly reduced cytopathic effect. Finally, the
third family is constituted of mycolactone A/B modified at C20
by polar groups (3a−c) and exhibits a significant cytotoxicity
with a LC50 between 15 and 50 nM, quite similar to the one of
the natural toxin (LC50 = 12 nM), thus highlighting that the
northern fragment can indeed be functionalized and also that a
complete southern fragment is important for the cytotoxicity.
In 2011, we reported a first generation synthesis of C8-
desmethyl mycolactone A/B analogues that led to a partial
structure−activity relationship by targeting especifically four
families (Figure 2B).33 The first one contains the C8-desmethyl
analogues whose C5-hydroxy group is either free (4a) or
esterified with five different C1′−C16′ fatty acid side chains
(4b−f), with 4b corresponding to the fatty acid side chain of
natural mycolactone A/B.34 The second class of analogues
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Scheme 2. Synthesis of Monodeoxy and Dideoxy Southern Fragments of Mycolactone Analogues
comprises two C14−C20 truncated analogues in which the C5-
hydroxy group is either free (5a) or esterified with the natural
C1′−C16′ southern fragment of mycolactone A/B (5b). The
third class includes a single ester of the polyenic fragment (6a),
whereas the fourth class encompasses the fluorescent derivative
7 used for in vitro imaging. We demonstrated that the less
potent derivatives were 5a and 6a and that suppression of the
southern (4a) or northern fragment (5b) of C8-desmethyl
mycolactone A/B led to a significant drop in cytopathic activity.
The importance of the absolute configurations of the
C12′,C13′,C15′-stereocluster (mycolactone numbering) was
also stressed out, since the more potent analogues possessed
the natural (12′S,13′S) configurations (as in 4b) whereas the
C15′-configuration was shown to be less crucial. In line with
the known cytopathic activity of mycolactone C (the C12′-
deoxy mycolactone A/B, LC50 = 186 nM),2,30 analogue 4e led
to 49% of cell rounding at 10 μM. Further studies on the more
potent analogue 4b uncovered that the lowest concentration
inducing 90% of cell rounding was 5 μM, to be compared with
the 40 nM found in our laboratory for the natural mycolactone
A/B. Consistent with the different activities reported for the
nine naturally occurring mycolactones (that differ only by the
structure of this southern fragment),2 this first set of data
clearly pointed toward the importance of the C1′−C16′
southern fragment for biological activity.
To gain further insight into structure−activity relationships,
we synthesized novel analogues of mycolactone including
additional variations in the southern chain and examined their
individual capacity to bind WASP and N-WASP in vitro. We
conclude that mycolactone binds and destabilizes the auto-
inhibited fold of WASP and N-WASP primarily via its lactone
core and southern chain.
RESULTS AND DISCUSSION
■
We first focused on the southern fragment and introduced
systematic variations in the C12′,C13′,C15′-stereocluster (both
from a substitution and configuration point of view) as well as
aromatic moieties in the polyenic motif.
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Scheme 3. Inclusion of Aromatic Moities in the Southern Fragment of Mycolactone Analogues
Variation of the C12′,C13′,C15′-Stereocluster. The
C12′,C13′,C15′-stereocluster is a crucial region of mycolac-
tones, as shown in an early study by Small7 and more recently
by Altmann and Pluschke31 and us.33 Besides the synthesis of
the syn−syn cluster present in the natural product, an efficient
access to all possible isomers has important implications in
terms of SAR studies. We specifically targeted the anti−syn
(18a and ent-18a), anti−anti (18b and ent-18b), and syn−anti
(18c) C12′,C13′,C15′-stereotriads using a catalytic asymmetric
approach to set up the three stereocenters (Scheme 1, General
Reaction Scheme). Starting from β-silyloxyaldehyde 8, (Z)-α,β-
unsaturated ester 9 was obtained as a single isomer because of
an Ando olefination reaction.35,36 Dihydroxylation37 of the
latter using commercially available AD-mix α led to compound
10a in a 90:10 diastereomeric ratio, in which the C12′,C13′-
stereocenters (mycolactone numbering) possessed the anti
relative configuration. Further chain elongation delivered the
(E,E)-dienylstannane 15a which is then cross-coupled with
iodotrienoate 16 using Liebeskind’s copper(I) thiophenecar-
boxylate38−40 as a mediator, at room temperature in NMP,
following our first generation synthesis.33 Although the yield
over two steps is moderate, this cross-coupling features an
active, stable, and economic mediator. A last saponification
finally delivered the desired C1′-C16′ southern fragment anti−
syn 18a. The same synthetic strategy was used for the
preparation of anti−anti 18b (with AD-mix β) and syn−anti
18c (Scheme 1, eq 1), switching the Ando olefination for a
classical Horner−Wadsworth−Emmons reaction. Eventually,
starting from β-silyloxyester ent-9, the last two complementary
southern fragments anti−anti ent-18b and anti−syn ent-18a
were obtained (Scheme 1, eq 2).
bis-methoxyphenyl esters according to the method of
Riguera.41−43 Following the previous route, ester 20 was
converted to the desired C15′-deoxy southern fragment 18d.
On the other hand, ent-20 was transformed to the desired
carboxylic acid ent-18d.
Finally, the excision of two hydroxyl groups from the
southern fragment was studied (Scheme 2, eqs 3−5). Several
synthetic routes leading to dideoxy-C13′,C15′ 18e were
evaluated, and only a Jacobsen hydrolytic kinetic resolution44,45
(HKR) of commercially available epoxide rac-21 proved
successful (Scheme 2, eq 3). By use of a catalytic amount of
22 (0.05 mol %), this HKR delivered on a multigram scale diol
23 in excellent yield and enantiomeric excess. Conversion of
the latter to α,β-unsaturated ester 24 and further to the desired
dideoxy-C13′,C15′ fragment 18e proceeded smoothly.
Finally, the last two dideoxy fragments 18f and 18g were
prepared in several steps starting from previous reaction
intermediates, diol 20 and α,β-unsaturated ester (E)-9,
respectively (Scheme 2, eqs 4 and 5). It should be noted that
all the southern fragments 18 are synthesized as a dynamic
mixture of C4′-C5′ isomers.46
Inclusion of Aromatic Moieties in the Southern
Fragment. As evocated in the previous section, the southern
fragment of the natural toxin exists as a dynamic mixture of
C4′-C5′ isomers, the Z-isomer being predominant because of
allylic strain between the C4′- and C6′-methyl groups in the
corresponding E-isomer.2 From a structure−activity relation-
ship point of view, a series of analogues deprived of this allylic
strain would be of interest by giving insights in the biological
activities of stereodefined isomers of the pentaenic fragment.
We thus embarked in the synthesis of three southern fragments
(Scheme 3, 18h−j) in which an aromatic motif would replace a
portion of the polyenic fragment. Three structural modulations
were targeted with the replacement of the C1′−C7′ fragment
with a benzoic (18h) or a cinnamic acid (18i) and the
replacement of the C1′−C4′ fragment with a benzoic acid
(18j). The syntheses relied on the Stille cross-coupling
reactions of aryl bromides 27 and 28 with the appropriate
vinylstannanes followed by ester hydrolysis (Scheme 3, eqs 1−
3).
On the basis of the structure of mycolactone C,2,30 it was
relevant to investigate structural modulation of the southern
fragment in the deoxy series. In addition to our previous
analogue 4e, two new derivatives were specifically targeted, 18d
and ent-18d, featuring a deoxy-C15′ position (Scheme 2, eqs 1
and 2). An asymmetric dihydroxylation of the more electron
rich double bond of the dienoate (E,E)-19 using both pseudo-
enantiomers of AD-mix led to the C15′-deoxy fragment 20 and
ent-20 as single enantiomers. The enantiomeric excess and
absolute configuration of 20 were proven by conversion to the
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Figure 3. Synthesis of analogues 5b−r via esterification of 5a with southern fragments 18 and ent-18.
Final Crafting of the Mycolactone Analogues. Having
in hand a set of 13 new C1′−C16′ southern fragments, we next
turn our attention to their esterification with a simplified core
structure of mycolactone. As shown in Figure 3, Yamaguchi
conditions followed by global deprotection with tetrabutylam-
monium fluoride delivered the desired new variants 5c−r.
Simplification of the Core Structure and Inclusion of
Fluorescent Motifs. Another set of two analogues (6b and
30) was obtained by esterification of 18k, the natural southern
fragment of mycolactone A/B (protected as a tri-TBS ether at
C12′, C13′, and C15′), with cyclohexanol (Scheme 4, eq 1)
and with the saturated mycolactone core, obtained in four steps
from the first generation synthesis intermediate 29 (Scheme 4,
eq 2).
The fluorescent analogue of mycolactone 7 (Figure 2) was
helpful in previous studies to assess the colocalization of
mycolactone with WASP in T lymphocytes.14 On the basis of
the known first generation intermediates 31a and 31b, five
C13-triazolomycolactone analogues were prepared by [3 + 2]
cycloadditions with the appropriate alkynes in moderate to
good yields (Scheme 4, eq 3), leading to the generation of
novel fluorescent derivatives (32a and 32d). The red-
fluorescing 32d may provide a useful alternative to green-
fluorescing 7 in multicolor imaging studies. Since 32a only
differs from 7 by the absence of the southern fragment, it
represents a valuable tool to investigate the role of this chain in
mycolactone diffusion in vivo. As shown in Figure 4, 32a and 7
both penetrated epithelial cells with saturable kinetics, reaching
a plateau after 24 h of incubation. Consistent with passive
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Scheme 4. Simplification of the Core Structure and Inclusion of Fluorescent Motifs
diffusion,47 the time-dependent accumulation of 32a in the cell
cytoplasm was faster than that of 7.
results were obtained with N-WASP (data no shown). No
binding could be detected with the 5a and 4a analogues, which
correspond to the simplified core structure of mycolactone A/B
bound or not to the northern chain (Figure 5A). In striking
contrast, mycolactone core linked to southern chain (5b)
bound WASP comparably to mycolactone (IC50 = 21.5 0.7
μM, Figure 6). In comparison, a cyclohexyl ester of the
southern fragment of mycolactone A/B (6b) was far less
reactive (IC50 = 135 21.2 μM).
We have previously shown that removal of the C8-methyl
group of mycolactone A/B, as in 4b, led to a 125-fold decrease
in cytopathic activity compared to the natural toxin.33 The
present results demonstrate that 4b is only 3-fold less efficient
for the binding to WASP, with an IC50 of 97.5 7.1 μM vs 32.3
22.1 μM for natural mycolactone A/B (Figure 5B and Figure
Mycolactone Analogues Bind to WASP and N-WASP.
As previously described, binding of natural mycolactone A/B to
the 502 amino acids protein WASP involves a minimal linear
domain of 114 amino acids (200−313) and binding to N-
WASP involves at least 91 amino acids (186−276) out of the
505 of the protein.14 Two interaction sites have been found, the
first one downstream of the CRIB region (Cdc42/Rac-
interactive-binding, 14 amino acids in WASP and N-WASP
(238−251 and 203−216, respectively)) and the second one in
the basic region (lysine-rich portion, 12 and 15 amino acids in
WASP and N-WASP, respectively (224−235 and 186−200)).
The binding of all mycolactone analogues to WASP and N-
WASP was assessed through their capacity to displace C12′-
biotinylated mycolactone A/B (the synthesis of which was
reported previously)14 from the mycolactone binding domains
(MBD) of WASP or N-WASP in ELISA.
6). Compared to 5b (IC50 = 21.5
0.7 μM), addition of a
(triazol-4-yl)ethanol to the core in northern position (32c)
limited binding (IC50 = 170 μM), as did the selective
epimerization of C12′ and C15′ (5i, IC50 = 100 μM) or the
removal of two hydroxyl groups of the southern fragment
The results obtained with the WASP MBD are shown in
Figures 5 and 6, as expressed by the half maximal inhibitory
concentrations relative to natural mycolactone A/B, which
displayed an IC50 of 32.3 22.1 μM (Figure 6). Comparable
(C13′,C15′-dideoxy 5m, IC50 = 350
70.7 μM) or one
hydroxyl group in C15′ (5k, IC50 = 250 0.7 μM). Saturation
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in the induction of a dose-dependent release of VCA from
immobilized MBD.14 Similar results were obtained with the
MBD in N-WASP (data no shown).
Since mycolactone-mediated activation of N-WASP causes
the detachment-induced death of epithelial cells,14 we then
compared mycolactone and 5b for capacity to inhibit cell
adhesion. We used the xCELLigence System (Roche Applied
Science), as it allows the dynamic monitoring of cell adhesion
through the measurement of electrical impedance across
interdigitated microelectrodes integrated on the bottom of
tissue culture E-Plates. Like mycolactone, 5b reduced the
adhesion capacity of HeLa cells (Figure 7B). In contrast, the
mycolactone analogue 5a, which is unable to bind WASP, did
not alter cell adhesion at the same concentration (data not
shown). Of note, 5b induced cell retraction at concentrations
>300 times higher than those of mycolactone (8 μM vs 26
nM), suggesting that the synthetic compound is less able to
gain access to N-WASP in the intracellular environment.
Figure 4. Diffusion of fluorescent mycolactone derivatives into
epithelial cells. The kinetics of intracellular accumulation of 32a and
7 in HeLa cells are shown. HeLa cells were seeded in 96-well plates
(104 cells/well) and incubated at 37 °C for 24 h prior to the addition
of fluorescent derivatives or vehicle as control. Cells were rinsed three
times with phosphate buffered saline (PBS) before reading of the
fluorescence on a microplate reader. Data are mean intensities of
fluorescence of treated cells per well (λexcitation = 490 nm; λemission = 520
nm) relative to controls, as measured in duplicate. They are
representative of two independent experiments.
CONCLUSION
■
In the present study, we report the synthesis of 23 new
analogues of mycolactone A/B together with their capacity to
bind WASP/N-WASP. A subset of 14 simplified analogues
missing the northern polyketide chain demonstrated a binding
potency equivalent to that of the natural toxin. Notably, the one
bearing mycolactone A/B southern chain also showed capacity
to activate WASP/N-WASP in vitro and induced cytopathic
effects. In addition to identification of the structural basis of
cytotoxicity in mycolactone, our study thus paves the way for
mechanistic studies of the mycolactone/WASP complex using a
simplified analogue as a surrogate of the natural exotoxin.
Unraveling the role of WASP/N-WASP in mycolactone-
induced immune suppression not only will help better
understand the pathogenesis of BU disease but also will
advance our understanding of the dialogue between the
cytoskeleton of immune cells and their effector functions.
Resolving the structure of the mycolactone/WASP complex
should generate insights into the physical mechanism of WASP
activation by mycolactone. It may help identify domains of N-
WASP/WASP that bind mycolactone without interfering with
the function of endogenous proteins, of interest to neutralize
the activity of this major virulence factor of M. ulcerans.
of the C8−C9 π-system (30) or introduction of a meta-
disubstituted aromatic motif on the C1′-carbonyl (5p, 5q) was
less detrimental to binding, with IC50 in the 60−70 μM range
(Figure 5). Notably, 14 other analogues demonstrated an
efficient binding to the MBD of WASP, with IC50 in the 10−44
μM range (Figure 6). These included the (hetero)aromatic
derivatives 32b (35
7.1 μM) and 5r (35
7.1 μM), the
C12′,C15′-dideoxy 5n (23 μM), the C12′-deoxy 5j (28 5.7
μM), and the C15′-deoxy 5l (27 1.4 μM). When the three
hydroxyl groups were present in C12′, C13′, and C15′ (5b−h),
IC50 values comparable to that of mycolactone A/B were
observed.
Overall, these investigations demonstrated that (1) an
efficient binding to the MBD of WASP was observed even
with mycolactone analogues lacking the northern fragment and
the C8-methyl group, one of the best representative example
being 5b, which is as potent as natural mycolactone; (2) the
relative and absolute configurations of the C12′, C13′, and
C15′ stereocenters can be modified individually without any
impact on the binding, as in 5c−h; (3) when two stereocenters
are inverted simultaneously, a sharp decrease in binding is only
observed for 5i (100 μM) which corresponds to the inversion
of the C12′,C15′ stereocenters; (4) one or two hydroxyl groups
can be selectively excised at the C12′, C13′, and/or at the C15′
positions (except for compounds 5k,m,i); (5) eventually, an
alkyl-substituted triazole is tolerated in the northern fragment
(32b) and an aromatic ring can be embedded in the southern
fragment when connected to the C3′ position (5r).
EXPERIMENTAL SECTION
■
General Procedures. NMR spectra were recorded on Bruker AV
300 or AV 400 spectrometer and calibrated using undeuterated solvent
as internal reference, unless otherwise indicated. Coupling constants
(J) were reported in hertz. Attached proton tests (APTs) were
performed to distinguish between different carbons in the 13C NMR
spectra. The following abbreviations were used to explain multi-
plicities: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet,
and b = broad. Optical rotations were recorded on a PerkinElmer
polarimeter (model 341LC) and are expressed in deg·cm2·g−1 units.
High-resolution mass spectra were recorded on an Agilent Q-TOF
(ESI) coupled with an Agilent 1100 series HPLC instrument. All
reactions were carried out in oven-dried glassware under an argon
atmosphere using dry solvents, unless otherwise noted. Tetrahydrofur-
an (THF) was distilled under argon from sodium benzophenone.
Toluene was dried using the Dry Solvent Station GT S100 developed
by Glass Technology, and dichloromethane was distilled over CaH2.
All other anhydrous solvents were purchased from Sigma-Aldrich.
Reagents were purchased from Aldrich, Acros, or Alfa Aesar and used
without further purification, unless otherwise noted. Yields refer to
chromatographically and spectroscopically (1H NMR) homogeneous
materials, unless otherwise noted. Reactions were monitored by thin-
5b Mimics Mycolactone for WASP Activation and
Alteration of Cell Adhesion. Having demonstrated that
mycolactone activates WASP by destabilizing the autoinhibited
fold of the protein,14 we examined the impact of the closest
structural analogue of the natural toxin, 5b, on the maintenance
of intramolecular interactions. A glutathione S-transferase
(GST) fused version of the MBD in WASP was immobilized
on gluthatione beads and used to capture WASP VCA in
solution (Figure 7A). The resulting complex was incubated
with 5b and dissociation monitored by electrophoretic analysis
of pulled down products. Strikingly, 5b mimicked mycolactone
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Figure 5. Mycolactone analogues binding WASP with IC50 in the 1000−50 μM range. Data are mean IC50 SD relative to vehicle controls, as
a
measured in replicate (n ≥ 2). Footnote letter in the figure indicates the following: Single determination.
layer chromatography (TLC) carried out on Merck TLC silica gel 60
F254 glass-coated plates, using UV light, iodine vapor, potassium
permanganate as visualizing agents. All separations were performed by
flash chromatography on Merck silica gel 60 (40−63 μm) unless
otherwise specified. All compounds used in biological assays were
7.75 mmol, 75%) as a pale yellow oil. 1H NMR (CDCl3, 300 MHz) δ
6.35 (dt, J = 11.6, 6.3 Hz, 1H), 5.84 (dt, J = 11.6, 1.7 Hz, 1H), 4.17 (q,
J = 7.1 Hz, 2H), 3.96 (dqd, J = 6.6, 6.1, 5.0 Hz, 1H), 2.88−5.68 (2H),
1.29 (t, J = 7.1 Hz, 3H), 1.16 (d, J = 6.1 Hz, 3H), 0.88 (s, 9H), 0.06 (s,
3H), 0.05 (s, 3H). 13C NMR (CDCl3, 100 MHz) δ 166.4, 146.9,
120.7, 67.9, 59.8, 38.6, 25.8 (3C), 23.7, 18.0, 14.2, −4.5, −4.8. HRMS-
ESI calculated for C14H29O3Si: m/z 273.1886 ([M + H]+). Found: m/
1
analyzed by H and 13C NMR and conformed to purities of ≥95%.
(S,Z)-Ethyl 5-((tert-Butyldimethylsilyl)oxy)hex-2-enoate ((Z)-
9). To a stirred solution of (o-Tol)2P(O)CH2CO2Et 35,36 (4.70 g,
13.50 mmol, 1.3 equiv) in dry THF (105 mL), under a nitrogen
atmosphere, was added NaI (1.56 g, 10.40 mmol) at 0 °C. After 5 min,
NaH (60% dispersion, 540 mg, 13.50 mmol, 1.3 equiv) was added.
The resulting solution was cooled to −78 °C, and 810 (2.10 g, 10.40
mmol) was added dropwise. After stirring 3 h at −78 °C, a saturated
aqueous NH4Cl solution was added and the reaction mixture was
extracted three times with Et2O. The combined organic extracts were
washed with brine, dried over MgSO4, filtered, and concentrated under
reduced pressure. The crude product was purified by flash
chromatography (cyclohexane/EtOAc 9:1) to afford (Z)-9 (2.11 g,
z 273.1881 ([M + H]+). [α]20 +12.9 (c 1.4, CHCl3).
D
(2S,3S,5S)-Ethyl 5-((tert-Butyldimethylsilyl)oxy)-2,3-
dihydroxyhexanoate (10a). To a stirred solution of AD-mix α
(5.06 g, 0.4% osmium, 1% (DHQ)2PHAL) in a mixture 1:1 t-BuOH/
H2O (30:30 mL) were added successively methanesulfonamide (300
mg, 3.15 mmol 0.9 equiv), potassium osmate(VI) dihydrate (10 mg,
27.14 μmol, 0.6 mol %), and (DHQ)2PHAL (114 mg, 0.15 mmol, 4
mol %). The mixture was stirred at room temperature until two clear
phases were produced. The solution was cooled to 0 °C, whereupon
the inorganic salts partially precipitate, and (Z)-9 (1.00 g, 3.68 mmol)
was then added. After stirring 48 h at 0 °C, Na2SO3 (9.2 g) was added
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Figure 6. Mycolactone analogues binding WASP with IC50 in the 50−10 μM range. Data are mean IC50 SD relative to vehicle controls, as
a
measured in replicate (n ≥ 2). Footnote letter in the figure indicates the following: Single determination.
and the reaction mixture was stirred for 30 min at room temperature.
The mixture was extracted three times with EtOAc. The combined
organic extracts were washed with an aqueous KOH solution (2 M),
brine, dried over MgSO4, filtered, and concentrated under reduced
pressure. The crude product was purified by chromatography
(cyclohexane/EtOAc 8:2) to give an inseparable mixture of
diastereoisomers (700 mg, 2.29 mmol, 62%, 90:10 in favor of the
desired diastereoisomer 10a) as a colorless oil. 1H NMR (CDCl3, 300
MHz) δ 4.34−4.18 (5H), 3.29 (br s, 1H), 3.00 (br s, 1H), 1.80 (m,
1H), 1.40 (m, 1H), 1.32 (t, J = 7.1 Hz, 3H), 1.22 (d, J = 6.2 Hz, 3H),
0.89 (s, 9H), 0.10 (s, 3H), 0.09 (s, 3H). 13C NMR (CDCl3, 100 MHz)
δ 172.6, 74.3, 70.1, 66.5, 61.8, 39.3, 25.8 (3C), 23.1, 17.9, 14.2, −4.5,
−5.1. HRMS-ESI calculated for C14H31O5Si: m/z 307.1941 ([M +
H]+). Found: m/z 307.1940 ([M + H]+).
(2R,3S,5S)-2,3,5-Tris((tert-butyldimethylsilyl)oxy)hexan-1-ol
(11). To a stirred solution of 10a (700 mg, 2.29 mmol) in dry DMF
(23 mL), under a nitrogen atmosphere, were added tert-butyldime-
thylsilyl chloride (2.07 g, 13.70 mmol, 6 equiv), imidazole (622 mg,
9.15 mmol, 4 equiv), and DMAP (56 mg, 0.46 mmol, 0.2 equiv). The
reaction mixture was stirred at room temperature for 48 h. The
resulting solution was hydrolyzed with water, and the aqueous layer
was extracted three times with a mixture of cyclohexane and DCM
(9:1). The combined organic extracts were washed with water, brine,
dried over MgSO4, filtered, and concentrated under reduced pressure.
The crude product was purified by chromatography (cyclohexane/
EtOAc 20:1) to give an inseparable mixture of diastereoisomeric esters
(766 mg, 1.43 mmol, 63%, 90:10 diastereomeric ratio) as a colorless
oil. 1H NMR (CDCl3, 300 MHz) δ 4.15 (q, J = 7.1 Hz, 2H), 4.15 (d, J
= 2.3 Hz, 1H), 4.07 (ddd, J = 7.5, 4.3, 2.3 Hz, 1H), 3.89 (m, 1H), 1.79
(ddd, J = 14.1, 7.8, 4.1 Hz, 1H), 1.59 (ddd, J = 14.1, 7.6, 4.6 Hz, 1H),
1.28 (t, J = 7.1 Hz, 3H), 1.16 (d, J = 6.1 Hz, 3H), 0.92 (s, 9H), 0.88 (s,
9H), 0.87 (s, 9H), 0.11 (s, 3H), 0.09 (s, 3H), 0.08 (s, 3H), 0.07 (s,
3H), 0.06 (s, 3H), 0.05 (s, 3H). 13C NMR (CDCl3, 100 MHz) δ
171.7, 77.5, 73.3, 65.8, 60.5, 44.6, 25.93 (3C), 25.90 (3C), 25.8 (3C),
24.6, 18.4, 18.1, 18.0, 14.2, −3.5, −4.0, −4.3, −4.6, −4.9, −5.3. HRMS-
ESI calculated for C26H58O5NaSi3: m/z 557.3490 ([M + Na]+).
Found: m/z 557.3492 ([M + Na]+).
To a stirred solution of the latter compound (766 mg, 1.43 mmol)
in dry DCM (12 mL), under a nitrogen atmosphere, was added
DIBAL-H (2.2 mL, 1.5 M in toluene, 3.15 mmol, 2.2 equiv) at 0 °C.
The reaction mixture was stirred at 0 °C for 1 h. Saturated aqueous
Rochelle salt solution was then added, and the resulting mixture was
warmed to room temperature and vigorously stirred overnight. The
aqueous layer was extracted with Et2O, and the combined organic
extracts were washed with water, brine, dried over MgSO4, filtered, and
concentrated under reduced pressure. The crude alcohol was purified
by chromatography (cyclohexane/EtOAc 95:5) to afford 11 (511 mg,
1.04 mmol, 73%) as the major diastereoisomer and 11b as the minor
1
one (51 mg, 0.10 mmol, 7%). H NMR (CDCl3, 300 MHz) δ 3.97−
3.90 (2H), 3.73 (dd, J = 12.5, 6.6 Hz, 1H), 3.63−3.57 (2H), 1.69
(ddd, J = 14.2, 8.3, 3.8 Hz, 1H), 1.50 (ddd, J = 14.2, 7.5, 3.6 Hz, 1H),
1.17 (d, J = 6.2 Hz, 3H), 0.92 (s, 9H), 0.89 (s, 9H), 0.88 (s, 9H), 0.13
(s, 3H), 0.11 (s, 3H), 0.10 (s, 3H), 0.10 (s, 3H), 0.08 (s, 6H). 13C
NMR (CDCl3, 100 MHz) δ 76.4, 73.6, 66.2, 63.7, 45.5, 26.0 (3C),
25.94 (3C), 25.93 (3C), 24.9, 18.3, 18.2, 18.0, −3.4, −3.9, −4.2, −4.3,
−4.5, −4.6. HRMS-ESI calculated for C24H57O4Si3: m/z 493.3565 ([M
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Figure 7. Effects of 5b on WASP activation and epithelial cell adhesion. (A) Dose-dependent displacement of WASP MBD-bound VCA by 5b: silver
staining of WASP MBD and VCA, after incubation of GST-fused WASP MBD (immobilized on glutathione-sepharose beads) with VCA in the
presence of increasing amounts of 5b, and analysis of bead-bound products by gel electrophoresis (left). The percentage of binding of VCA (right) is
calculated as a function of the concentration of mycolactone A/B and 5b, relative to controls. (B) Reduction of epithelial cell adhesion upon addition
of 5b. HeLa cells were left to adhere for 4 h at 37 °C prior to the addition of mycolactone A/B (26 nM), 5b (8 and 16 μM), or DMSO as vehicle
control (Ctrl) and monitoring of impedance for 24 h. Data are mean cell index, calculated in quadruplicate.
+ H]+). Found: m/z 493.3561 ([M + H]+). [α]20 +9.7 (c 0.9,
CHCl3).
ESI calculated for C29H62O5NaSi3: m/z 597.3803 ([M + Na]+).
D
Found: m/z 597.3807([M + Na]+). [α]20 +4.4 (c 0.4, CHCl3).
D
(1E,3E)-(5R,6S,8S)-Tris(tert-butyldimethylsilyloxy)-1-iodo-3-
methylnona-1,3-diene (14). To a stirred solution of 13 (375 mg,
0.65 mmol) in dry DCM (4.0 mL), under a nitrogen atmosphere, was
added DIBAL-H (0.95 mL, 1.5 M in toluene, 1.43 mmol, 2.2 equiv) at
0 °C. The reaction mixture was stirred at 0 °C for 1 h. Saturated
aqueous Rochelle salt solution was then added and the resulting
mixture warmed to room temperature and vigorously stirred overnight.
The aqueous layer was extracted with Et2O, and the combined organic
extracts were washed with water, brine, dried over MgSO4, filtered and
concentrated under reduced pressure. The crude reacion mixture was
purified by chromatography (cyclohexane/EtOAc 9:1) to afford
(4R,5S,7S,E)-4,5,7-tris((tert-butyldimethylsilyl)oxy)-2-methyloct-2-en-
(2S,3S,5S)-2,3,5-Tris((tert-butyldimethylsilyl)oxy)hexanal
(12). To a stirred solution of 11 (500 mg, 1.01 mmol) in dry DCM
(2.0 mL), under a nitrogen atmosphere, were added iodosobenzene
diacetate (360 mg, 1.12 mmol, 1.1 equiv) and TEMPO (16 mg, 0.10
mmol, 0.1 equiv) at 0 °C. The reaction mixture was stirred at room
temperature for 5 h. Water was then added to the reaction mixture
which was extracted three times with DCM. The combined organic
extracts were washed with water, brine, dried over MgSO4, filtered, and
concentrated under reduced pressure. The crude aldehyde 12 was used
1
in the next step without further purification. H NMR (CDCl3, 300
MHz) δ 9.62 (d, J = 1.9 Hz, 1H), 4.08 (m, 1H), 3.91 (ddd, J = 7.9, 6.1,
4.2 Hz, 1H), 3.87 (dd ap.t, J = 2.0 Hz, 1H), 1.75 (ddd, J = 14.1, 7.9, 4.3
Hz, 1H), 1.61 (ddd, J = 14.1, 7.2, 4.2 Hz, 1H), 1.17 (d, J = 6.1 Hz,
3H), 0.94 (s, 9H), 0.89 (s, 9H), 0.87 (s, 9H), 0.13 (s, 3H), 0.11 (s,
6H), 0.08 (s, 9H).
1
1-ol (338 mg, 0.63 mmol, 97%) as a colorless oil. H NMR (CDCl3,
300 MHz) δ 5.45 (dq, J = 8.9, 1.2 Hz, 1H), 4.27 (dd, J = 8.8, 2.6 Hz,
1H), 4.02 (s, 2H), 3.96 (m, 1H), 3.83 (dt, J = 7.7, 2.8 Hz, 1H), 1.71
(d, J = 1.2 Hz, 3H), 1.54 (ddd, J = 14.1, 8.6, 3.1 Hz, 1H), 1.37 (ddd, J
= 14.1, 7.9, 3.4 Hz, 1H), 1.15 (d, J = 6.2 Hz, 3H), 0.89 (s, 9H), 0.89 (s,
9H), 0.88 (s, 9H), 0.11 (s, 3H), 0.09 (s, 3H), 0.07 (s, 6H), 0.04 (s,
3H), 0.01 (s, 3H). 13C NMR (CDCl3, 100 MHz) δ 135.4, 126.6, 74.7,
73.7, 68.7, 66.2, 44.7, 26.1 (3C), 26.0 (3C), 25.9 (3C), 25.0, 18.3, 18.2,
18.0, 14.5, −3.2, −3.5, −4.1, −4.3, −4.5, −4.6. HRMS-ESI calculated
for C27H60O4NaSi3: m/z 555.3697 ([M + Na]+). Found: m/z
(4R,5S,7S,E)-Ethyl 4,5,7-Tris((tert-butyldimethylsilyl)oxy)-2-
methyloct-2-enoate (13). To a stirred solution of crude aldehyde
12 in 1,2-dichloroethane (5 mL), under a nitrogen atmosphere, was
added (1-ethoxycarbonylethylidene)triphenylphosphorane (550 mg,
1.50 mmol, 1.5 equiv) at room temperature. The reaction mixture was
warmed to 70 °C and stirred for 48 h. The resulting solution was then
cooled to room temperature. The solvent was removed under reduced
pressure and the residue purified by chromatography (cyclohexane/
EtOAc 95:5) to give 13 (375 mg, 0.65 mmol, 65% for two steps) as a
colorless oil. 1H NMR (CDCl3, 300 MHz) δ 6.69 (dq, J = 9.1, 1.4 Hz,
1H), 4.32 (dd, J = 9.2, 2.8 Hz, 1H), 4.20 (q, J = 7.1 Hz, 2H), 3.96 (m,
1H), 3.88 (dt, J = 7.8, 2.9 Hz, 1H), 1.87 (d, J = 1.4 Hz, 3H), 1.56 (ddd,
J = 14.1, 8.6, 3.1 Hz, 1H), 1.38 (ddd, J = 14.1, 8.3, 3.8 Hz, 1H), 1.30 (t,
J = 7.1 Hz, 3H), 1.15 (d, J = 6.2 Hz, 3H), 0.89 (s, 18H), 0.87 (s, 9H),
0.12 (s, 3H), 0.09 (s, 3H), 0.07 (s, 3H), 0.06 (s, 3H), 0.04 (s, 3H),
0.01 (s, 3H). 13C NMR (CDCl3, 100 MHz) δ 167.9, 141.8, 127.4,
74.4, 74.3, 66.0, 60.6, 44.8, 26.0 (3C), 25.92 (3C), 25.88 (3C), 24.9,
18.3, 18.2, 18.0, 14.2, 13.4, −3.3, −3.7, −4.2, −4.3, −4.5, −4.8. HRMS-
555.3699 ([M + Na]+). [α]20 +12.3 (c 1.0, CHCl3).
D
To a stirred solution of the latter alcohol (338 mg, 0.63 mmol) in
dry DCM (6.3 mL) under a nitrogen atmosphere was added MnO2
(830 mg, 9.52 mmol, 15 equiv) at room temperature. The reaction
mixture was warmed to 40 °C and vigorously stirred for 48 h. The
resulting mixture was cooled to room temperature and filtered through
Celite with DCM. The solvent was then removed under reduced
pressure to give (4R,5S,7S,E)-4,5,7-tris((tert-butyldimethylsilyl)oxy)-2-
methyloct-2-enal (265 mg, 0.50 mmol, 79%) as a colorless oil which
was used in the next step without further purification. 1H NMR
(CDCl3, 300 MHz) δ 9.45 (s, 1H), 6.44 (dq, J = 8.9, 1.4 Hz, 1H), 4.49
(dd, J = 8.9, 2.6 Hz, 1H), 3.99−3.93 (2H), 1.80 (d, J = 1.4 Hz, 3H),
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1.51 (ddd, J = 14.1, 8.9, 3.0 Hz, 1H), 1.36 (ddd, J = 14.1, 7.9, 3.0 Hz,
1H), 1.15 (d, J = 6.2 Hz, 3H), 0.89 (s, 18H), 0.87 (s, 9H), 0.14 (s,
3H), 0.11 (s, 3H), 0.07 (s, 6H), 0.05 (s, 3H), 0.01 (s, 3H).
NH4Cl solution, the mixture was extracted three times with EtOAc
and the combined organic extracts were washed with brine, dried over
MgSO4, filtered, and concentrated under reduced pressure. The crude
material was purified by preparative TLC (cyclohexane/EtOAc 7:3) to
afford 18a (72 mg, 0.11 mmol, 64%) as a yellow oil. NMR analysis
showed the presence of mixture of isomers 4′E/4′Z = 85:15. 1H NMR
(acetone-d6, 300 MHz, 4′E isomer) δ 7.93 (d, J = 15.5 Hz, 1H, Z
isomer), 7.35 (d, J = 15.6 Hz, 1H, E isomer), 6.67 (dd, J = 15.1, 11.1
Hz, 1H), 6.46 (d, J = 15.1 Hz, 1H), 6.46 (s, 1H), 6.39 (d, J = 11.1 Hz,
1H), 5.87 (d, J = 15.6 Hz, 1H), 5.63 (d, J = 9.1 Hz, 1H), 4.49 (dd, J =
9.1, 2.8 Hz, 1H), 4.06 (m, 1H), 3.97 (dt, J = 7.6, 2.9 Hz, 1H), 2.09 (s,
3H), 2.06 (s, 3H), 1.93 (s, 3H), 1.64 (ddd, J = 14.1, 8.8, 3.1 Hz, 1H),
1.41 (ddd, J = 14.1, 7.7, 3.2 Hz, 1H), 1.17 (d, J = 6.2 Hz, 3H), 0.91 (s,
18H), 0.89 (s, 9H), 0.17 (s, 3H), 0.15 (s, 3H), 0.11 (s, 3H), 0.10 (s,
3H), 0.09 (s, 3H), 0.05 (s, 3H). 13C NMR (acetone-d6, 100 MHz, 4′E
isomer) δ 168.1, 151.6, 144.3, 140.3, 136.2, 135.5, 135.4, 135.0, 133.3,
125.3, 117.3, 75.9, 75.2, 67.1, 45.8, 26.6 (3C), 26.5 (3C), 26.4 (3C),
25.5, 19.0, 18.9, 18.7, 17.2, 14.4, 13.9, −2.9, −3.0, −3.7, −3.8, −4.1,
−4.3. HRMS-ESI calculated for C37H70O5NaSi3: m/z 701.4423 ([M +
Na]+). Found: m/z 701.4423 ([M + Na]+).
(6S,7S,9E,12R)-7-Methyl-2-oxo-12-(propan-2-yl)-1-oxacyclo-
dodec-9-en-6-yl-(2E,4E,6E,8E,10E,12R,13S,15S)-12,13,15-trihy-
droxy-4,6,10-trimethylhexadeca-2,4,6,8,10-pentaenoate (5e).
To a solution of 18a (22 mg, 0.03 mmol) in benzene (0.4 mL)
were added diisopropylethylamine (23 μL, 0.12 mmol, 6 equiv), 2,4,6-
trichlorobenzoyl chloride (11 μL, 0.06 mmol, 3 equiv), and DMAP
(20 mg). The reaction mixture was stirred at room temperature for 15
min, and 5a33 (5.5 mg, 0.02 mmol) was added. After being stirred at
room temperature for 14 h, an aqueous saturated solution of sodium
hydrogenocarbonate was added to the reaction mixture. The aqueous
layer was extracted three times with benzene. The combined organic
layers were washed with brine, dried over MgSO4, filtered, and
concentrated under reduced pressure. The crude product was purified
on preparative TLC, eluting with heptane/ethyl acetate 90:10 to give
(6S,7S,9E,12R)-7-methyl-2-oxo-12-(propan-2-yl)-1-oxacyclododec-9-
en-6-yl (2E,4E,6E,8E,10E,12R,13S,15S)-12,13,15-tris[(tert-
butyldimethylsilyl)oxy]-4,6,10-trimethylhexadeca-2,4,6,8,10-pentae-
noate (13 mg, 0.014 mmol, 71%) as a yellow oil. A (4′E)/(4′Z) =
61:39 mixture could be detected by 1H NMR analysis. 1H NMR
(CDCl3, 300 MHz) δ 7.93 (d, J = 15.5 Hz, 1H), Z isomer), 7.36 (d, J =
15.5 Hz, 1H, E isomer), 6.51 (dd, J = 14.8, 11.8 Hz, 1H), 6.37 (d, J =
14.8 Hz, 1H), 6.34 (s, 1H), 6.26 (d, J = 11.8 Hz, 1H), 5.85 (d, J = 15.5
Hz, 1H), 5.56 (d, J = 9.0 Hz, 1H), 5.51 (m, 1H), 5.25 (m, 1H), 4.82−
4.73 (2H), 4.34 (dd, J = 2.8, 8.8 Hz, 1H), 3.94 (m, 1H), 3.86 (td, J =
2.8, 7.7 Hz, 1H), 2.49 (m, 1H), 2.30 (m, 1H), 2.14−2.06 (2H), 2.05
(s, 3H), 2.02 (s, 3H), 1.95 (m, 1H), 1.84 (s, 3H), 1.87−1.61 (6H),
1.13 (d, J = 6.1 Hz, 3H), 0.94 (s, 3H), 0.93 (s, 3H), 0.91 (s, 3H), 0.90
(s, 3H), 0.88 (s, 9H), 0.87 (s, 9H), 0.86 (s, 9H), 0.10 (s, 3H), 0.08 (s,
3H), 0.05 (s, 6H), 0.02 (s, 3H), −0.01 (s, 3H).
To a stirred solution of CrCl2 (368 mg, 3.00 mmol, 6 equiv) in dry
THF (3.0 mL), under a nitrogen atmosphere, was added a solution of
(4R,5S,7S,E)-4,5,7-tris((tert-butyldimethylsilyl)oxy)-2-methyloct-2-
enal (265 mg, 0.50 mmol) and CHI3 (590 mg, 1.49 mmol, 3 equiv) in
dry THF (2.1 mL) dropwise. The reaction mixture was stirred at room
temperature for 12 h. The resulting mixture was hydrolyzed with water
and diluted with Et2O. The aqueous layer was extracted three times
with Et2O and the combined organic extracts were washed with water,
brine, dried over MgSO4, filtered, and concentrated under reduced
pressure. The crude product was then purified by chromatography
(cyclohexane/toluene 100:1) to afford 14 (250 mg, 0.38 mmol, 75%)
as a pale yellow oil. 1H NMR (C6D6, 300 MHz) δ 7.05 (d, J = 14.1 Hz,
1H), 6.01 (d, J = 14.1 Hz, 1H), 5.56 (dq, J = 9.1, 1.2 Hz, 1H), 4.40
(dd, J = 9.1, 2.8 Hz, 1H), 4.12 (m, 1H), 4.05 (dt, J = 7.8, 2.8 Hz, 1H),
1.69 (ddd, J = 14.1, 8.9, 2.8 Hz, 1H), 1.57 (ddd, J = 14.1, 7.9, 2.9 Hz,
1H), 1.47 (d, J = 1.2 Hz, 3H), 1.13 (d, J = 6.2 Hz, 3H), 1.03 (s, 9H),
1.00 (s, 18H), 0.25 (s, 3H), 0.23 (s, 3H), 0.14 (s, 3H), 0.11 (s, 3H),
0.10 (s, 3H), 0.06 (s, 3H). 13C NMR (C6D6, 100 MHz) δ 149.6, 135.0,
134.6, 76.6, 75.4, 74.7, 66.9, 45.7, 26.7 (3C), 26.6 (3C), 26.5 (3C),
25.6, 19.0, 18.9, 18.7, 13.1, −2.6, −2.8, −3.6, −3.7, −3.9, −4.1.
(1E,3E)-(5R,6S,8S)-Tris(tert-butyldimethylsilyloxy)-1-tributyl-
stannyl-3-methylnona-1,3-diene (15a). To a stirred solution of 14
(250 mg, 0.38 mmol) in dry Et2O (2.0 mL), under a nitrogen
atmosphere, was added n-BuLi (0.17 mL, 2 M in hexane, 0.57 mmol,
1.5 equiv) at −78 °C. The reaction mixture was stirred for 20 min at
−78 °C, and Bu3SnCl (0.17 mL, 0.57 mmol, 1.5 equiv) was added.
The resulting solution was stirred at −78 °C for another 20 min and
allowed to warm to room temperature for 1 h. Saturated aqueous
NaHCO3 solution was then added, and the aqueous layer was
extracted three times with Et2O. The combined organic extracts were
washed with brine, dried over MgSO4, filtered, and concentrated under
reduced pressure. The crude 15a was used in the next step without
further purification.
(2E,4E,6E,8E,10E,12R,13S,15S)-Ethyl 12,13,15-Tris((tert-
butyldimethylsilyl)oxy)-4,6,10-trimethylhexadeca-2,4,6,8,10-
pentaenoate (17). To a stirred solution of crude 15a and tetra-n-
butylammonium diphenylphosphinate (415 mg, 0.89 mmol, 2.3 equiv)
in dry NMP (2.7 mL), under a nitrogen atmosphere, was added 0.2
mL of a solution of 1633 (240 mg, 0.77 mmol, 2 equiv) in NMP (1.8
mL). After addition of copper thiophenecarboxylate (150 mg, 0.77
mmol, 2 equiv), the rest of the 16 solution was added dropwise, and
the resulting mixture was stirred at room temperature for 40 min. The
reaction mixture was then diluted with Et2O and filtered through
neutral alumina oxide. The filtrate was washed with water, brine, dried
over MgSO4, filtered, and concentrated under reduced pressure. The
crude product was then purified by preparative TLC (cyclohexane/
EtOAc 9:1) to afford 17 (120 mg, 0.17 mmol, 45% for two steps) as a
yellow oil. NMR analysis showed the presence of a mixture of isomers
4′E/4′Z = 93:7. 1H NMR (CDCl3, 300 MHz, 4′E isomer) δ 7.95 (d, J
= 15.3 Hz, 1H, Z isomer), 7.38 (d, J = 15.5 Hz, 1H, E isomer), 6.50
(dd, J = 14.9, 11.1 Hz, 1H), 6.37 (d, J = 14.9 Hz, 1H), 6.36 (s, 1H),
6.27 (d, J = 11.1 Hz, 1H), 5.87 (d, J = 15.5 Hz, 1H), 5.57 (d, J = 9.1
Hz, 1H), 4.35 (dd, J = 9.1, 2.8 Hz, 1H), 4.23 (q, J = 7.1 Hz, 2H), 3.96
(m, 1H), 3.87 (dt, J = 7.6, 2.9 Hz, 1H), 2.06 (s, 3H), 2.03 (s, 3H), 1.85
(s, 3H), 1.53 (m, 1H), 1.36 (m, 1H), 1.32 (t, J = 7.1 Hz, 3H), 1.14 (d,
J = 6.1 Hz, 3H), 0.89 (s, 9H), 0.88 (s, 9H), 0.87 (s, 9H), 0.11 (s, 3H),
0.09 (s, 3H), 0.08 (s, 3H), 0.06 (s, 3H), 0.03 (s, 3H), 0.00 (s, 3H). 13C
NMR (CDCl3, 100 MHz, 4′E isomer) δ 167.5, 150.7, 143.7, 139.7,
135.1, 134.7, 134.0, 133.9, 132.2, 123.7, 116.1, 74.8, 74.1, 66.2, 60.2,
44.9, 26.1 (3C), 26.0 (3C), 25.9 (3C), 25.0, 18.33, 18.27, 18.0, 17.1,
14.3, 14.2, 13.4, −3.3, −3.5, −4.2, −4.3, −4.6, −4.7.
To a solution of the latter compound (13 mg, 0.015 mmol) in THF
(0.1 mL) was added TBAF (0.130 mmol, 1 M in THF, 9 equiv), and
the solution was stirred at room temperature for 4 h. CaCO3 (40 mg),
Dowex 50WX8-400 (110 mg), and MeOH (0.3 mL) were added, and
the reaction mixture was stirred for 1 h. After being filtered and
concentrated under reduced pressure, the crude product was purified
by preparative TLC (CH2Cl2/MeOH 90:10) to give 5e (6 mg, 0.01
mmol, 67%). A (4′E)/(4′Z) = 56:44 mixture could be detected by 1H
1
NMR analysis. H NMR (acetone-d6, 400 MHz) δ 7.93 (d, J = 15.6
Hz, 1H, Z isomer), 7.37 (d, J = 15.5 Hz, 1H, E isomer), 6.63 (dd, J =
11.1, 15.0 Hz, 1H), 6.47 (s, 1H), 6.36 (d, J = 15.0 Hz, 1H), 6.17 (d, J =
11.1 Hz, 1H), 5.89 (d, J = 15.5 Hz, 1H), 5.68 (d, J = 8.5 Hz, 1H), 5.50
(m, 1H), 5.30 (m, 1H), 4.78−4.67 (2H), 4.38 (m, 1H), 4.04 (m, 1H),
3.88 (m, 1H), 3.81 (m, 1H), 3.69 (m, 1H), 3.63 (m, 1H), 2.49 (m,
1H), 2.32 (m, 1H), 2.12−2.08 (2H), 2.06 (s, 3H), 1.97 (s, 3H), 1.95−
1.92 (2H), 1.90 (s, 3H), 1.80−1.75 (4H), 1.70−1.62 (2H), 1.54−1.49
(2H), 1.13 (d, J = 6.1 Hz, 3H), 0.92 (s, 3H), 0.90 (s, 3H), 0.87 (s,
3H). 13C NMR (acetone-d6, 100 MHz) δ 173.9, 167.8, 152.2, 144.1,
141.6, 137.3, 136.5, 136.1, 134.1, 133.0, 128.0, 125.8, 120.6, 118.4,
80.2, 77.3, 73.5, 73.3, 65.9, 43.1, 39.8, 37.9, 37.0, 36.1, 34.1, 25.6, 22.0,
21.6, 20.9, 19.9, 19.4, 18.1, 15.3, 14.3. HRMS-ESI calculated for
(2E,4E,6E,8E,10E,12R,13S,15S)-12,13,15-Tris((tert-
butyldimethylsilyl)oxy)-4,6,10-trimethylhexadeca-2,4,6,8,10-
pentaenoic Acid (18a). To a stirred solution of 17 (117 mg, 0.16
mmol) in a mixture THF/MeOH/H2O (3.5/0.9/0.9 mL) was added
LiOH (40 mg, 1.65 mmol, 10 equiv). The resulting mixture was stirred
at room temperature for 18 h. After addition of a saturated aqueous
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C34H52O7Na: m/z 595.3605 ([M + Na]+). Found: m/z 595.3600 ([M
+ Na]+).
the University of Haute-Alsace, and the University of
Strasbourg.
Biological Procedures. Natural Mycolactones. Mycolactone
(A/B form) was purified from M. ulcerans 1615 (ATCC 35840).7 A
biologically active, biotin-bearing derivative of mycolactone A/B was
prepared as described.14 The concentration and purity of all
mycolactone preparations were assessed by HPLC−MS/MS.48 Stock
solutions in dimethyl sulfoxide (DMSO) were conserved at −20 °C,
protected from light. They were diluted >1000 times in culture
medium extemporaneously and compared to vehicle-treated controls
in bioassays.
WASP/N-WASP Constructs. GST-fused MBDs corresponding to
the MBD in WASP (200−313) or N-WASP (186−276) and His6-
tagged VCA constructs were expressed and purified as described.14
ELISAs. The binding of each mycolactone variant to WASP/N-
WASP was measured indirectly, through their capacity to displace
biotinylated mycolactone from immobilized MBDs.14 Briefly, GST-
fused MBDs (5 μg/mL) in PBS were coated onto plastic 96 microtiter
wells (NUNC MaxiSorp) by overnight incubation at 4 °C. After a 1 h
blocking step at room temperature with PBS + 1% bovine serum
albumin (BSA), plates were washed with PBS + 0.05% Tween20 and
incubated for 2 h at room temperature with biotinylated mycolactone
(1 μM) plus increasing amounts of mycolactone variants (0−10 μM)
in PBS + 0.1% BSA. After washing, complexes were revealed by
addition of streptavidin-conjugated alkaline phosphatase (1 h, room
temperature) and colorimetric detection.
VCA Displacement Assay. The capacity of 5b to displace VCA
from the MBD in WASP or N-WASP was adapted from the procedure
used to indicate mycolactone dissociating activity.14 Briefly, GST-fused
MBD was immobilized on glutathione-sepharose beads and then
incubated with His6-tagged VCA in the presence of increasing
amounts of 5b. After thorough washing of the beads, bound products
were analyzed by gel electrophoresis and silver staining.
Epithelial Cell Adhesion Assay. Background of the E-plates was
determined in 50 μL cell culture medium prior to addition of 150 μL
of HeLa cell suspension (20 000 cells/well). Plates were then placed
into the real-time cell analyzer (RTCA) station, and impedance was
measured every 15 min. Impedance values are represented by cell
index (CI) values ((Zi − Z0) [Ω]/15 [Ω]; Z0 is background resistance,
and Zi is individual time point resistance).
ABBREVIATIONS USED
■
Arp, actin-related protein; AT2R, angiotensin II receptor; CPE,
cytopathic effect; CRIB, Cdc42/Rac-interactive-binding;
CuTC, copper thiophenecarboxylate; HKR, hydrolytic kinetic
resolution; MBD, mycolactone binding domain; N-WASP,
neural Wiskott−Aldrich syndrome protein; TEMPO, 2,2,6,6-
tetramethylpiperidine 1-oxyl; VCA, verprolin-cofilin-acidic;
WASP, Wiskott−Aldrich syndrome protein
REFERENCES
■
(1) O’Brien, D. P.; Comte, E.; Serafini, M.; Ehounou, G.; Antierens,
A.; Vuagnat, H.; Christinet, V.; Hamani, M. D.; du Cros, P. The urgent
need for clinical, diagnostic, and operational research for management
of Buruli ulcer in Africa. Lancet Infect. Dis. 2014, 14, 435−440.
(2) Chany, A.-C.; Tresse, C.; Casarotto, V.; Blanchard, N. History,
biology and chemistry of Mycobacterium ulcerans infections (Buruli
ulcer disease). Nat. Prod. Rep. 2013, 30, 1527−1567.
(3) Walsh, D. S.; Portaels, F.; Meyers, W. M. Buruli ulcer
(Mycobacterium ulcerans infection): a re-emerging disease. Clin.
Microbiol. Newsl. 2009, 31, 119−127.
(4) Demangel, C.; Stinear, T. P.; Cole, S. T. Buruli ulcer: reductive
evolution enhances pathogenicity of Mycobacterium ulcerans. Nat. Rev.
Microbiol. 2009, 7, 50−60.
(5) Hong, H.; Demangel, C.; Pidot, S. J.; Leadlay, P. F.; Stinear, T.
Mycolactones: immunosuppressive and cytotoxic polyketides pro-
duced by aquatic mycobacteria. Nat. Prod. Rep. 2008, 25, 447−454.
(6) MacCallum, P.; Tolhurst, J. C.; Buckle, G.; Sissons, H. A. A new
mycobacterial infection in man. J. Pathol. Bacteriol. 1948, 60, 93−122.
(7) George, K. M.; Chatterjee, D.; Gunawardana, G.; Welty, D.;
Hayman, J.; Lee, R.; Small, P. L. C. Mycolactone: a polyketide toxin
from Mycobacterium ulcerans required for virulence. Science 1999, 283,
854−857.
(8) Gunawardana, G.; Chatterjee, D.; George, K. M.; Brennan, P.;
Whittern, D.; Small, P. L. C. Characterization of novel macrolide
toxins, mycolactones A and B, from a human pathogen, Mycobacterium
ulcerans. J. Am. Chem. Soc. 1999, 121, 6092−6093.
(9) Kishi, Y. Chemistry of mycolactones, the causative toxins of
Buruli ulcer. Proc. Natl. Acad. Sci. U.S.A. 2011, 108, 6703−6708.
(10) Song, F. B.; Fidanze, S.; Benowitz, A. B.; Kishi, Y. Total
synthesis of the mycolactones. Org. Lett. 2002, 4, 647−650.
(11) Fidanze, S.; Song, F. B.; Szlosek-Pinaud, M.; Small, P. L. C.;
Kishi, Y. Complete structure of the mycolactones. J. Am. Chem. Soc.
2001, 123, 10117−10118.
ASSOCIATED CONTENT
* Supporting Information
Experimental procedures and NMR spectra. This material is
available free of charge via the Internet at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Authors
*A.-C. C.: phone, +33 3 8933 6855; fax, +33 3 8933 6860; e-
mail, chanyac@gmail.com.
■
(12) Benowitz, A. B.; Fidanze, S.; Small, P. L. C.; Kishi, Y.
Stereochemistry of the core structure of the mycolactones. J. Am.
Chem. Soc. 2001, 123, 5128−5129.
*N. B.: phone, +33 3 8933 6824; fax, +33 3 8933 6860; e-mail,
n.blanchard@unistra.fr.
́ ́
(13) Guenin-Mace, L.; Oldenburg, R.; Chretien, F.; Demangel, C.
Pathogenesis of skin ulcers: lessons from the Mycobacterium ulcerans
and Leishmania spp. pathogens. Cell. Mol. Life Sci. 2014, 71, 2443−
2450.
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
́
(14) Guenin-Mace, L.; Veyron-Churlet, R.; Thoulouze, M. I.; Romet-
Lemonne, G.; Hong, H.; Leadlay, P. F.; Danckaert, A.; Ruf, M. T.;
Mostowy, S.; Zurzolo, C.; Bousso, P.; Chretien, F.; Carlier, M. F.;
Demangel, C. Mycolactone activation of Wiskott−Aldrich syndrome
proteins underpins Buruli ulcer formation. J. Clin. Invest. 2013, 123,
1501−1512.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
(15) Thrasher, A. J.; Burns, S. O. WASP: a key immunological
multitasker. Nat. Rev. Immunol. 2010, 10, 182−192.
(16) Boulkroun, S.; Guenin-Mace, L.; Thoulouze, M. I.; Monot, M.;
́
Merckx, A.; Langsley, G.; Bismuth, G.; Di Bartolo, V.; Demangel, C.
Mycolactone suppresses T cell responsiveness by altering both early
signaling and posttranslational events. J. Immunol. 2010, 184, 1436−
1444.
We thank Sandrine Vitry (Institut Pasteur, Paris, France) for
helping us with the XCelligence assays of epithelial cell
adhesion. This work received financial support from the
“Association Raoul Follereau”, the “Fondation pour la
Recherche Me
DEQ20120323704”, the “Region Ile de France”, the CNRS,
́
dicale, Equipes FRM 2012, Project
7394
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Article
(17) Coutanceau, E.; Decalf, J.; Martino, A.; Babon, A.; Winter, N.;
Cole, S. T.; Albert, M. L.; Demangel, C. Selective suppression of
dendritic cell functions by Mycobacterium ulcerans toxin mycolactone. J.
Exp. Med. 2007, 204, 1395−1403.
up of 40% of the L929 cells after 48 h (versus 100% of cell rounding
with 4b), demonstrating the relative importance of the C15′-position.
(35) Ando, K. Highly selective synthesis of Z-unsaturated esters by
using new Horner−Emmons reagents, ethyl (diarylphosphono)-
acetates. J. Org. Chem. 1997, 62, 1934−1939.
(18) Coutanceau, E.; Marsollier, L.; Brosch, R.; Perret, E.; Goossens,
P.; Tanguy, M.; Cole, S. T.; Small, P. L. C.; Demangel, C. Modulation
of the host immune response by a transient intracellular stage of
Mycobacterium ulcerans: the contribution of endogenous mycolactone
toxin. Cell Microbiol. 2005, 7, 1187−1196.
(36) Ando, K. Practical synthesis of Z-unsaturated esters by using a
new Horner−Emmons reagent, ethyl diphenylphosphonoacetate.
Tetrahedron Lett. 1995, 36, 4105−4108.
(37) Kolb, H. C.; VanNieuwenhze, M. S.; Sharpless, K. B. Catalytic
asymmetric dihydroxylation. Chem. Rev. 1994, 94, 2483−2547.
(38) Allred, G. D.; Liebeskind, L. S. Copper-mediated cross-coupling
of organostannanes with organic iodides at or below room
temperature. J. Am. Chem. Soc. 1996, 118, 2748−2749.
́
(19) Guenin-Mace, L.; Carrette, F.; Asperti-Boursin, F.; Le Bon, A.;
Caleechurn, L.; Di Bartolo, V.; Fontanet, A.; Bismuth, G.; Demangel,
C. Mycolactone impairs T cell homing by suppressing microRNA
control of L-selectin expression. Proc. Natl. Acad. Sci. U.S.A. 2011, 108,
12833−12838.
(39) Donnard, M.; Blanchard, N. Copper-catalyzed C−C bond
formation in natural product synthesis: elegant and efficient solutions
to a key bond disconnection. In Copper-Mediated Cross-Coupling
Reactions; John Wiley & Sons, Inc.: Hoboken, NJ, 2013; pp 683−723.
(40) Evano, G.; Blanchard, N.; Toumi, M. Copper-mediated coupling
reactions and their applications in natural products and designed
biomolecules synthesis. Chem. Rev. 2008, 108, 3054−3131.
(20) Hall, B.; Simmonds, R. Pleiotropic molecular effects of the
Mycobacterium ulcerans virulence factor mycolactone underlying the
cell death and immunosuppression seen in Buruli ulcer. Biochem. Soc.
Trans. 2014, 42, 177−183.
(21) Hall, B. S.; Hill, K.; McKenna, M.; Ogbechi, J.; High, S.; Willis,
A. E.; Simmonds, R. E. The pathogenic mechanism of the
Mycobacterium ulcerans virulence factor, mycolactone, depends on
blockade of protein translocation into the ER. PLoS Pathog. 2014, 10,
e1004061.
(22) Pahlevan, A. A.; Wright, D. J. M.; Andrews, C.; George, K. M.;
Small, P. L. C.; Foxwell, B. M. The inhibitory action of Mycobacterium
ulcerans soluble factor on monocyte/T cell cytokine production and
NF-kappa B function. J. Immunol. 1999, 163, 3928−3935.
(23) Simmonds, R. E.; Lali, F. V.; Smallie, T.; Small, P. L. C.;
Foxwell, B. M. Mycolactone inhibits monocyte cytokine production by
a posttranscriptional mechanism. J. Immunol. 2009, 182, 2194−2202.
(24) Marion, E.; Song, O.-R.; Christophe, T.; Babonneau, J.;
Fenistein, D.; Eyer, J.; Letournel, F.; Henrion, D.; Clere, N.; Paille,
́
(41) Seco, J. M.; Quinoa, E.; Riguera, R. Assignment of the absolute
̃
configuration of polyfunctional compounds by NMR using chiral
derivatizing agents. Chem. Rev. 2012, 112, 4603−4641.
́
(42) Freire, F.; Seco, J. M.; Quinoa, E.; Riguera, R. Determining the
̃
absolute stereochemistry of secondary/secondary diols by 1H NMR:
basis and applications. J. Org. Chem. 2005, 70, 3778−3790.
́
(43) Seco, J. M.; Quinoa, E.; Riguera, R. The assignment of absolute
̃
configuration by NMR. Chem. Rev. 2004, 104, 17−118.
(44) Ford, D. D.; Nielsen, L. P. C.; Zuend, S. J.; Musgrave, C. B.;
Jacobsen, E. N. Mechanistic basis for high stereoselectivity and broad
substrate scope in the (salen)Co(III)-catalyzed hydrolytic kinetic
resolution. J. Am. Chem. Soc. 2013, 135, 15595−15608.
(45) Tokunaga, M.; Larrow, J. F.; Kakiuchi, F.; Jacobsen, E. N.
Asymmetric catalysis with water: efficient kinetic resolution of terminal
epoxides by means of catalytic hydrolysis. Science 1997, 277, 936−938.
(46) Even if stereoisomerically pure southern fragments could be
prepared, Negishi has shown that spontaneous isomerization occurred
upon deprotection of the C12′,C13′,C15′ stereotriad; see the
following: Wang, G. W.; Yin, N.; Negishi, E. Highly stereoselective
total synthesis of fully hydroxy-protected mycolactones A and B and
their stereoisomerization upon deprotection. Chem.Eur. J. 2011, 17,
4118−4130.
(47) Snyder, D. S.; Small, P. L. C. Uptake and cellular actions of
mycolactone, a virulence determinant for Mycobacterium ulcerans.
Microb. Pathog. 2003, 34, 91−101.
(48) Hong, H.; Coutanceau, E.; Leclerc, M.; Caleechurn, L.; Leadlay,
P. F.; Demangel, C. Mycolactone diffuses from Mycobacterium
ulcerans-infected tissues and targets mononuclear cells in peripheral
blood and lymphoid organs. PLoS Neglected Trop. Dis. 2008, 2, e325.
́ ́
V.; Guerineau, N. C.; Saint Andre, J.-P.; Gersbach, P.; Altmann, K.-H.;
Stinear, T. P.; Comoglio, Y.; Sandoz, G.; Preisser, L.; Delneste, Y.;
Yeramian, E.; Marsollier, L.; Brodin, P. Mycobacterial toxin induces
analgesia in Buruli ulcer by targeting the angiotensin pathways. Cell
2014, 157, 1565−1576.
(25) Danser, A. H. J.; Anand, P. The angiotensin II type 2 receptor
for pain control. Cell 2014, 157, 1504−1506.
(26) Hande, S. M.; Kazumi, Y.; Lai, W. G.; Jackson, K. L.; Maeda, S.;
Kishi, Y. Synthesis and structure of two new mycolactones isolated
from M. ulcerans subsp. shinshuense. Org. Lett. 2012, 14, 4618−4621.
(27) Spangenberg, T.; Aubry, S.; Kishi, Y. Synthesis and structure
assignment of the minor metabolite arising from the frog pathogen
Mycobacterium liflandii. Tetrahedron Lett. 2010, 51, 1782−1785.
(28) Kim, H. J.; Kishi, Y. Total synthesis and stereochemistry of
mycolactone F. J. Am. Chem. Soc. 2008, 130, 1842−1843.
(29) Aubry, S.; Lee, R. E.; Mahrous, E. A.; Small, P. L. C.;
Beachboard, D.; Kishi, Y. Synthesis and structure of mycolactone E
isolated from frog Mycobacterium. Org. Lett. 2008, 10, 5385−5388.
(30) Judd, T. C.; Bischoff, A.; Kishi, Y.; Adusumilli, S.; Small, P. L. C.
Structure determination of mycolactone C via total synthesis. Org. Lett.
2004, 6, 4901−4904.
(31) Scherr, N.; Gersbach, P.; Dangy, J.-P.; Bomio, C.; Li, J.;
Altmann, K.-H.; Pluschke, G. Structure−activity relationship studies
on the macrolide exotoxin mycolactone of Mycobacterium ulcerans.
PLoS Neglected Trop. Dis. 2013, 7, e2143.
(32) Gersbach, P.; Jantsch, A.; Feyen, F.; Scherr, N.; Dangy, J.-P.;
Pluschke, G.; Altmann, K.-H. A ring-closing metathesis (RCM)-based
approach to mycolactones A/B. Chem.Eur. J. 2011, 17, 13017−
13031.
(33) Chany, A.-C.; Casarotto, V.; Schmitt, M.; Tarnus, C.; Guenin-
́
Mace, L.; Demangel, C.; Mirguet, O.; Eustache, J.; Blanchard, N. A
diverted total synthesis of mycolactone analogues: an insight into
Buruli ulcer toxins. Chem.Eur. J. 2011, 17, 14413−14419.
(34) Analogue 4f was recently synthesized in our group and for the
sake of clarity is directly compared in Figure 2 with 4a−e that we
reported in 2011. 4f at a concentration of 10 μM induced the rounding
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